The laboratory is new to the Kidney Disease Branch and the first year has mostly been focused on getting its physical space renovated, outfitted with equipment and staffed with personnel. We currently are staffed at 60% of capacity and expect to fill the final two slots within the next 4 monthswe have been awaiting completion of our microscope room/tissue culture facility, which should be done in late autumn 2010. We have also successfully transferred the labs biological reagents and mouse colony from Johns Hopkins to the intramural program at NIH. From a scientific perspective, we have made the following progress on our two themes: 1. Hypothesis-driven: a) PCP and PKD1: We have generated a series of new MDCK cell lines expressing EGFP-tagged PKD1 and used these to identify a novel localization pattern for PC1. We have shown co-localization with a sub-set of PCP-related proteins and that altering expression of the latter alters the activity and localization of PC1. b) PKHD1 and SMURFS: We had previously generated a series of MDCK cell lines with inducible expression of recombinant, multiply-tagged PKHD1 which we used to characterize the biochemical and cell biological properties of polyductin (PD), the PKHD1 gene product. In characterizing the cell lines, we found that PKHD1 expression resulted in changes in cellular morphology. In examining the mechanisms underlying these effects, we found that changes in PKHD1 expression results in changes in Rho family protein expression, altered SMURF1/2 distribution in cells, and enhanced TGF-beta signaling. Our current efforts are focused on demonstrating the in vivo relevance of these findings in collaboration with Dr. Kaimori, a former post-doctoral fellow. 2. Discovery-based: a) Systems-based discovery of pathways regulated by Pkd1: We had previously identified a two-day interval in mice that determines its susceptibility to acquired Pkd1 loss. Mice with Pkd1 inactivation acquired prior to post-natal d12 (P12) developed severe cystic disease within 5 days whereas inactivation at P14 or later did not result in cysts until 4-5 months later. Much of my current research effort is now focused on understanding what is going on during this developmental window, how this relates to PC1 function, and what accounts for the late onset of disease in the adult phase. We are pursuing this aim using a variety of approaches. We are analyzing serial changes in mRNA, microRNA, and protein expression from the time just after Pkd1 is inactivated through the time at which cysts first become apparent. We simultaneously are examining what transpires in normals during this timeframe, seeking to identify a regulatory network that governs this late maturation phase. Our studies this year have focused primarily on the rapid-onset forms of disease. We have identified several regulatory factors whose activity appears to be dysregulated shortly after Pkd1 inactivation, and we are presently verifying these results with independent methods.